The Mechanism Of Polyphenol From Millet Bran On Inhibiting Colitis And Colitis-associated Colorectal Cancer By Modulating Gut Microbiota

Inflammatory bowel disease(IBD)is a chronic,easily relapsing inflammatory disease of the digestive tract,which includes ulcerative colitis(UC)and Crohn’s disease(CD).Patients with IBD typically present with loss of appetite,abdominal pain,diarrhea,and blood in the stool.As the incidence of IBD has risen globally,it has been recognized drawn widespread attention from researchers.Colorectal cancer(CRC)is the third most common cancer in the world,and its mortality rate is second only to lung cancer and breast cancer among the causes of death in various human malignancies.IBD is thought to promote carcinogenesis and increase the risk of colitis-associated colorectal cancer(CAC)due to the effects of chronic inflammation.The pathogenesis of CAC involves many factors,including host genes,environment,diet habit,immune system and composition of commensal microorganisms.In the long-term evolution process,the intestinal commensal microbiome has formed a complex symbiotic ecosystem with the host gastrointestinal tract,which is the "second genome" acquired by the human body.In the occurrence and development of IBD and CAC,many factors,such as the composition,regulation and metabolites of gut microbiota,and the microbial microenvironment are closely related.Therefore,regulating gut microbiota is an effective strategy for the prevention and treatment of colitis and CAC.Polyphenols,one type of small molecular compounds,are widely present in a variety of plants such as tea,soybeans and vegetables,and belong to secondary metabolites in plants.Studies have reported that polyphenols possess an abundance of physiological activities,such as anti-oxidation,anti-inflammation,anti-tumor,reducing blood lipids and preventing atherosclerosis.In addition,studies have also shown that polyphenols can be used as prebiotics to regulate the community of gut microbiota.In the previous research,we extracted bound polyphenol of inner shell from millet bran(BPIS)from foxtail millet bran.Cell and nude mouse experiments showed that BPIS has anti-inflammatory and anti-tumor activities.In the present study,we continued to explore the effect of BPIS on colitis and colitis-associated colon cancer in vivo and the regulation of BPIS microbiota.Main research results are listed as follow:(1)BPIS ameliorates DSS-induced colitis by remodeling gut microbiome.To explore the effect of BPIS on IBD,C57BL/6J mice were induced by dextran sodium sulfate(DSS)to construct experimental colitis model.Results showed that BPIS administration effectively relieved the weight loss,decreased disease active index(DAI)scores,restrained the secretion of pro-inflammatory cytokines.BPIS prevented gut barrier damage by enhancing tight junction proteins Claudin1,ZO-1 and Occludin,increasing the number of goblet cells and facilitating the gene expressions of mucin family.In addition,BPIS restored the gut microbiota composition and increased the relative abundance of Firmicutes,Lachnospiraceae and Rikenellaceae and restrained the growth of Bacteroidetes,S24-7 and Staphylococcaceae.Concentrations of short-chain-fatty acids(SCFAs)generated by gut microbiota were elevated in BPIS treated colitis mice.These data suggest that BPIS effectively ameliorates DSS-induced colitis by preventing intestinal barrier damage and promoting gut microbiota community.(2)BPIS inhibits inflammation by increasing the concentration of niacin.Through LC-MS/MS metabolomics analysis,it was found that BPIS affected the changes in the fecal metabolic profile of mice with colitis,and the concentration of niacin was significantly increased by BPIS.Enrichment analysis also showed that BPIS regulated pathways of nicotinate and nicotinamide metabolism.Cell experiments proved that niacin inhibited the inflammatory response of LPS-induced colonic cells reduced the secretion of inflammatory factors TNF-α and IL-1β.Furthermore,it showed that niacin inhibited the phosphorylation of AKT and NF-κB through the receptor GPR109A;when the expression of GPR109 A was interfered by si RNA,the inhibitory effects of niacin on phosphorylated AKT-NF-κB and high inflammatory factors were abolished.In addition,it was also found that phosphorylated AKT-NF-κB were remarkably suppressed by BPIS-treated colitis mice.These results suggest that BPIS exerts anti-inflammatory effects through niacinGPR109A-AKT-NF-κB axis.(3)Inhibitory Effects of BPIS on colitis-associated carcinogenesis by the restoration of gut microbiota.The effect of BPIS on AOM/DSS-induced colitisassociated colorectal cancer(CAC)mice was evaluated.The results showed that BPIS restored the colon length,reduced the incidence of tumors,inhibited the growth of tumors,and increased the survival rate of mice.BPIS decreased the concentration of inflammatory factors TNF-α and IL-1β in the colon,and significantly inhibited the expressions of cyclooxygenase 2(COX-2)and EGF module-containing mucin-like receptor 1(EMR1).Furthermore,Results showed that BPIS promoted cell apoptosis by inhibiting Bcl-2 and activating caspase3.BPIS suppressed cell proliferation marker proliferating cell nuclear antigen(PCNA).Analysis of the fecal microbial composition of CAC mice also found that BPIS remodeled the overall structure of the gut microbiota from tumor-bearing mice toward that of the normal counterparts,including two phyla Firmicutes and Bacteroidetes,and eight genera such as Prevotella,Desulfovibrio,Coprococcus,etc.(4)Coprococcus eutactus plays a key role in BPIS remodeling gut microbiota.Random forest analysis showed that Coprococcus was the key microorganism that mediates BPIS to regulate the microbiome of colitis mice and CAC mice.and the abundance of Coprococcus was significantly increased by BPIS.Human metagenome database GMrepo also indicated Coprococcus,in particular,Coprococcus eutactus(C.eutactus),was negatively associated with IBD.The q RT-PCR and fluorescence in situ hybridization(FISH)experiments found that the abundance of C.eutactus was significantly increased in BPIS-treated colitis mice,indicating that BPIS can promote growth of C.eutactus.(5)C.eutactus alleviates colitis via acetate-mediated Ig A secretion.In DSSinduced colitis model mice,administration with C.eutactus effectively relieve colitis,including restoring mouse body weight,reducing disease activity index(DAI),restoring colon length,inhibiting splenomegaly,and protecting intestinal epithelial structure.C.eutactus reduced the expression of inflammatory cytokines such as TNF-α and IL-1β,and increased the concentration of anti-inflammatory factors such as IL-10.In addition,C.eutactus enhanced the maturation of goblet cells and the expressions of mucins and restored the expressions of tight junction proteins.As a short-chain fatty acid-producing bacterium,C.eutactus mainly generates acetic acid.Interestingly,not only high levels of secretory immunoglobulin A(SIg A)but also increased Ig A-producing plasma cells were observed in colitis mice during the administration of C.eutactus.Importantly,our data demonstrated that colonic SIg A is specifically coated on pathogens of Enterobacteriaceae.Owing to the selective binding effect of SIg A on microorganisms,the microbial diversity in the intestinal lumen and mucosa of C.eutactus-treated colitis mice was significantly restored,and the microbiota structure was remodeled.